When a laundry detergent is used for washing laundry, the pH of the washing liquid is mostly from 10 to 11; i.e., within an alkaline range. Therefore, an enzyme to be incorporated into laundry detergents is required to exhibit an optimum pH in an alkaline region and to be stable under an alkaline condition.
Conventionally known alkaline cellulases which can be incorporated into laundry detergents or other detergents include alkaline cellulase derived from Bacillus sp. KSM-635 belonging to Bacillus (Japanese Patent Publication (kokoku) No. 60-23158, Japanese Patent Publication (kokoku) No. 6-030578, U.S. Pat. No. 4,945,053, etc.); alkaline cellulase derived from Bacillus sp. KSM-64 (Shikata et al. Agric. Biol. Chem., 54, 91-96, 1990, Sumitomo et al., Biosci. Biotechnol. Biochem., 56, 872-877, 1992); heat-resistant alkaline cellulase produced from mesophilic and alkaliphilic fungi, Bacillus sp. KSM-S237 (FERM-BP7875: deposited on Feb. 6, 1997 with Independent Administrative Institution of National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary (Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki, Japan (postal code 305-8566))) (Japanese Patent Application Laid-Open (kokai) No. 10-313859); alkaline cellulase derived from Bacillus sp. KSM-N257 (Japanese Patent Application No. 12-281378); and alkaline cellulase derived from Bacillus sp. KSM-N131 (Japanese Patent Application No. 12-373859). However, in the case where the substrate is carboxymethylcellulose (CMC) these alkaline cellulases exhibit an optimum reaction pH of about 9; therefore, the cellulases do not have the optimum pH under conditions encountered during laundry washing.
In the meanwhile, a study has been done on changing the optimum reaction pH of a glucosidase. The study shows that the optimum pH of a glucosidase is shifted from alkali to neutral by constructing a chimeric protein from an alkaline (NK1) cellulase derived from alkaliphilic Bacillus and neutral cellulase (BSC) derived from Bacillus subtilis (Park et al. Protein Enz. 6, 921-926, 1993).
Recently, it has been reported that the optimum pH of cellobiohydrolase (Cel7A) derived from Trichoderma reesei was increased as compared with that of its wild-type strain by substitution of amino acid residues in the vicinity of its active center (Beker et al, Biochem. J., 356, 19-31, 2001). However, in this case, the optimum pH of the wild-type enzyme falls within an acidic range, and the increase in the optimum pH of the mutant is within 1 pH unit or less.
Thus, there has been substantially no report in which the optimum reaction pH of glucosidase is shifted toward the alkaline side.
The present invention is directed to the provision of a mutated alkaline cellulase having an optimum pH as an enzyme to be incorporated into detergent, which is obtained by modifying the alkaline cellulase gene.